Cardiac protective techniques in left breast radiotherapy: rapid selection criteria for routine clinical decision making.

OBJECTIVE: In left breast radiotherapy (RT) desired heart doses may be achieved without heart-sparing RT techniques in some patients. We aimed to examine the existence of predictive factors and cutoff points to determine which patients are the main candidates for heart-sparing RT techniques. MATERIAL AND METHOD: Dosimetric data for left breast cancer was examined. RT plans were made at conventional doses to the breast and peripheral lymph nodes. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., IBM Corp., Armonk, NY). RESULT: 114 cases were evaluated by ROC (Receiver operating characteristic) analysis in the breast-conserving surgery (BCS) and mastectomy groups. While only left lung volume (AUC: 0.74, 95% CI 0.61-0.87, p = 0.002) was significant in BCS cases, in cases with mastectomy, left lung volume (AUC: 0.81, 95% CI 0.69-0.94, p = 0.002) and lung/heart volume ratio (AUC: 0.83, 95% CI 0.70-0.96, p = 0.001) had a significant relationship with the relevance of heart doses. The cutoff point of 1.92 was selected for the lung/heart volume ratio for the mastectomized patients. Moreover, the cutoff point 1154 cc and 1208 cc was determined for the left lung volume for the BCS and mastectomized patients, respectively. CONCLUSION: Various cutoff points in left breast RT can be used to predict whether RT plans will meet QUANTEC (Quantitative Analysis of Normal Tissue Effects in the Clinic) heart dose limits. Evaluating only these few cutoff points before planning makes it possible to eliminate 70% of patients with BCS and 40% of patients with mastectomy from respiratory-controlled methods, which require time and effort. Patients with lung volume and lung/heart volume ratio smaller than the cutoff values can be considered primary candidates for heart-sparing techniques.

Life science-based neuroscience education at large Western Public Universities.

The last 40 years have seen a remarkable increase in the teaching of neuroscience at the undergraduate level. From its origins as a component of anatomy or physiology departments to its current status as an independent interdisciplinary field, neuroscience has become the chosen field of study for many undergraduate students, particularly for those interested in medical school or graduate school in neuroscience or related fields. We examined how life science-based neuroscience education is offered at large public universities in the Western United States. By examining publicly available materials posted online, we found that neuroscience education may be offered as an independent program, or as a component of biological or physiological sciences at many institutions. Neuroscience programs offer a course of study involving a core series of courses and a collection of topical electives. Many programs provide the opportunity for independent research, or for laboratory-based training in neuroscience. Features of neuroscience programs at Western universities closely matched those seen at the top 25 public universities, as identified by U.S. News & World Report. While neuroscience programs were identified in many Western states, there were several states in which public universities appeared not to provide opportunities to major in neuroscience. © 2016 Wiley Periodicals, Inc.

Green tea polyphenols require the mitochondrial iron transporter, mitoferrin, for lifespan extension in Drosophila melanogaster.

Green tea has been found to increase the lifespan of various experimental animal models including the fruit fly, Drosophila melanogaster. High in polyphenolic content, green tea has been shown to reduce oxidative stress in part by its ability to bind free iron, a micronutrient that is both essential for and toxic to all living organisms. Due to green tea's iron-binding properties, we questioned whether green tea acts to increase the lifespan of the fruit fly by modulating iron regulators, specifically, mitoferrin, a mitochondrial iron transporter, and transferrin, found in the hemolymph of flies. Publicly available hypomorph mutants for these iron regulators were utilized to investigate the effect of green tea on lifespan and fertility. We identified that green tea could not increase the lifespan of mitoferrin mutants but did rescue the reduced male fertility phenotype. The effect of green tea on transferrin mutant lifespan and fertility were comparable to w1118 flies, as observed in our previous studies, in which green tea increased male fly lifespan and reduced male fertility. Expression levels in both w1118 flies and mutant flies, supplemented with green tea, showed an upregulation of mitoferrin but not transferrin. Total body and mitochondrial iron levels were significantly reduced by green tea supplementation in w1118 and mitoferrin mutants but not transferrin mutant flies. Our results demonstrate that green tea may act to increase the lifespan of Drosophila in part by the regulation of mitoferrin and reduction of mitochondrial iron.

Studying the pathophysiologic connection between cardiovascular and nervous systems using stem cells.

The cardiovascular and nervous systems are deeply connected during development, health, and disease. Both systems affect and regulate the development of each other during embryogenesis and the early postnatal period. Specialized neural crest cells contribute to cardiac structures, and a number of growth factors released from the cardiac tissue (e.g., glial cell line-derived neurotrophic factor, neurturin, nerve growth factor, Neurotrophin-3) ensure proper maturation of the incoming parasympathetic and sympathetic neurons. Physiologically, the cardiovascular and nervous systems operate in harmony to adapt to various physical and emotional conditions to maintain homeostasis through sympathetic and parasympathetic nervous systems. Moreover, neurocardiac regulation involves a neuroaxis consisting of cortex, amygdala, and other subcortical structures, which have the ability to modify lower-level neurons in the hierarchy. Given the interconnectivity of cardiac and neural systems, when one undergoes pathological changes, the other is affected to a certain extent. In addition, there are specific neurocardiac diseases that affect both systems simultaneously, such as Huntington disease, Lewy body diseases, Friedreich ataxia, congenital heart diseases, Danon disease, and Timothy syndrome. Over the last decade, in vitro modeling of neurocardiac diseases using induced pluripotent stem cells (iPSCs) has provided an invaluable opportunity to elevate our knowledge about the brain-heart connection, since previously primary cardiomyocytes and neurons had been extremely difficult to maintain long-term in vitro. Ultimately, the ability of iPSC technology to model abnormal functional phenotypes of human neurocardiac disorders, combined with the ease of therapeutic screening using this approach, will transform patient care through personalized medicine in the future. © 2016 Wiley Periodicals, Inc.

Extension of Drosophila Lifespan by Rhodiola rosea Depends on Dietary Carbohydrate and Caloric Content in a Simplified Diet.

The root and rhizome extract of Rhodiola rosea has been extensively used in traditional medicine to improve physical and mental performance and to protect against stress. We, and others, have reported that R. rosea can extend lifespan in flies, worms, and yeast. We also previously found that the extract can act independently of dietary restriction (DR), a treatment that can extend lifespan in a range of model organisms. In flies, DR is implemented through a reduction in dietary yeast content. Here, we report that the ability of R. rosea extract to extend lifespan in flies is dependent on the carbohydrate and caloric content when supplemented with a simplified diet composed of yeast and sucrose. R. rosea extract elevated the sugar content in flies and down-regulated hexokinase expression, suggesting that it perturbs carbohydrate metabolism in flies. In our previous studies, bananas, barley malt, and corn syrup provided dietary carbohydrates, and R. rosea extract could extend lifespan with a range of caloric levels. We conclude that the lifespan-extending effect of R. rosea extract in flies is dependent on dietary carbohydrate and caloric contents coupled with an interaction with complex dietary components present in bananas, barley, or corn.

The impact of green tea polyphenols on development and reproduction in Drosophila melanogaster.

Although, green tea has numerous health benefits, adverse effects with excessive consumption have been reported. Using Drosophila melanogaster, a decrease in male fertility with green tea was evidenced. Here, the extent of green tea toxicity on development and reproduction was investigated. Drosophila melanogaster embryos and larvae were exposed to various doses of green tea polyphenols (GTP). Larvae exposed to 10 mg/mL GTP were slower to develop, emerged smaller, and exhibited a dramatic decline in the number of emerged offspring. GTP protected flies against desiccation but sensitized them to starvation and heat stress. Female offspring exhibited a decline in reproductive output and decreased survival while males were unaffected. GTP had a negative impact on reproductive organs in both males and females (e.g., atrophic testes in males, absence of mature eggs in females). Collectively, the data show that high doses of GTP adversely affect development and reproduction of Drosophila melanogaster.

Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

Spatiotemporally different origins of NG2 progenitors produce cortical interneurons versus glia in the mammalian forebrain.

The studies on the exact lineage composition of NG2 expressing progenitors in the forebrain have been controversial. A number of studies have revealed the heterogeneous nature of postnatal NG2 cells. However, NG2 cells found in embryonic dates are far less understood. Our study indicates that early NG2 progenitors from a ventral origin (i.e., before embryonic day 16.5) tangentially migrate out of the medial ganglionic eminence and give rise to interneurons in deep layers of the dorsal cerebral cortex. The majority of myelinating oligodendrocytes found in both cortical gray and white matters are, in contrast, derived from NG2 progenitors with a neonatal subventricular zone origin. Our lineage tracing data reflect the heterogeneous nature of NG2 progenitor populations and define the relationship between lineage divergence and spatiotemporal origins. Beyond the typical lineage tracing studies of NG2(+) cells, by costaining with lineage-specific markers, our study addresses the origins of heterogeneity and its implications in the differentiation potentials of NG2(+) progenitors.

Epigenetic regulation of stem cells differentiating along the neural lineage.

Many lineage-specific genes are poised and silenced in stem cells. Upon differentiation, genes that are related to self-renewal and alternative lineages are stably silenced. CpG methylation at proximal promoters and PRC2-mediated H3K27me3 play a role in silencing genes temporarily or permanently, with or without coexistence of active epigenetic marks, respectively. Interestingly, DNA methylation on neuronal genes that is distal to transcription start site enable transcription activation owing to its ability to repel PRC2-mediated inhibition. In addition, DNA demethylase Tet proteins play a role in regulation of changes in DNA methylation and related H3K27me3 during differentiation. Collectively, a complex epigenetic network formed by H3K4me3, histone acetylation/deacetylation, H3K27me3 and DNA methylation/demethylation act together to regulate stem cell self-renewal and differentiation.

Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes.

DNA methylation at proximal promoters facilitates lineage restriction by silencing cell type-specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such nonproximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent nonproximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, nonpromoter DNA methylation by Dnmt3a may be used for maintaining active chromatin states of genes critical for development.

Transcriptional regulation of neuronal differentiation: the epigenetic layer of complexity.

The transcriptional programs of neural progenitor cells change dynamically during neurogenesis, a process regulated by both intrinsic and extrinsic factors. Although many of the transcription factors required for neuronal differentiation have long been identified, we are only at the brink of understanding how epigenetic mechanisms influence transcriptional activity and the accessibility of transcription factors to bind consensus cis-elements. Herein, we delineate the chief epigenetic modifications and the machinery responsible for these alterations. Further, we review the epigenetic modifications presently known to participate in the maintenance of the neural progenitor cell state and in the regulation of neuronal differentiation.

CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain.

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP(+) cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133(+). Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133(+) ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133(+) ependymal cells represent an additional-perhaps more quiescent-stem cell population in the mammalian forebrain.

Neurons or glia? Can SHP2 know it all?

Normal development of the nervous system relies on the spatially and temporally well-controlled differentiation of neurons and glia. Here, we discuss the intra- and extracellular molecular mechanisms that underlie the sequential genesis of neurons and glia, emphasizing recent studies describing the role of a signaling molecule, the tyrosine phosphatase SHP2, in normal brain development. Activation of SHP2 simultaneously enhances downstream activation of the MEK-ERK pathway, which subsequently promotes neurogenesis, while inhibiting the JAK-STAT pathway, which is critical for astroglial differentiation. Mutations in SHP2 that increase its tyrosine phosphatase activity cause a mental retardation-related disorder, Noonan syndrome. An imbalance in neurogenesis versus gliogenesis due to SHP2 mutations may contribute to Noonan syndrome.

Subventricular zone neuronal progenitors undergo multiple divisions and retract their processes prior to each cytokinesis.

Mitotically active progenitor cells from the anterior portion of the forebrain subventricular zone (SVZa), which give rise throughout life to olfactory bulb interneurons, bear processes and express neuronal markers. To understand how rodent SVZa neuronal progenitors coordinate division and process formation, we used time-lapse videomicroscopy to analyse the proliferative behavior of SVZa progenitors in dissociated cell culture continuously for up to five generations. The cell cycle time of these cultured SVZa cells assessed videomicroscopically (cytokinesis to cytokinesis) was similar to the cell cycle time along the rostral migratory stream in vivo (14-17 h). The relationship between process extension, process retraction and cytokinesis was assessed quantitatively for 120 cells undergoing cytokinesis. Although all of these cells had elaborated processes, virtually all of them completely withdrew their processes prior to cytokinesis. Process withdrawal was rapid and tightly coupled to cytokinesis; 50% of the cells studied initiated process retraction within 30 min of cytokinesis and 96% had begun to withdraw their processes within 60 min of cytokinesis. In SVZa progenitor cell lineages, the sequence of process extension, process retraction and division is repeated over multiple generations. This complete withdrawal of processes prior to division differentiates SVZa progenitor cells from the characteristics reported for several other process-bearing types of neural progenitor cells, including sympathetic neuroblasts, cerebral cortical radial glia, and cerebellar and retinal progenitors. Collectively, our findings indicate that SVZa progenitors employ different cellular mechanisms than other neural progenitors to regulate proliferation and differentiation.

Coupling of cell migration with neurogenesis by proneural bHLH factors.

After cell birth, almost all neurons in the mammalian central nervous system migrate. It is unclear whether and how cell migration is coupled with neurogenesis. Here we report that proneural basic helix-loop-helix (bHLH) transcription factors not only initiate neuronal differentiation but also potentiate cell migration. Mechanistically, proneural bHLH factors regulate the expression of genes critically involved in migration, including down-regulation of RhoA small GTPase and up-regulation of doublecortin and p35, which, in turn, modulate the actin and microtubule cytoskeleton assembly and enable newly generated neurons to migrate. In addition, we report that several DNA-binding-deficient proneural genes that fail to initiate neuronal differentiation still activate migration, whereas a different mutation of a proneural gene that causes a failure in initiating cell migration still leads to robust neuronal differentiation. Collectively, these data suggest that transcription programs for neurogenesis and migration are regulated by bHLH factors through partially distinct mechanisms.

A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis.

During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokine-induced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.

Intrinsic and extrinsic regulation of the proliferation and differentiation of cells in the rodent rostral migratory stream.

An overriding principle of development is that neurons become permanently postmitotic once they initiate differentiation. Work in our laboratory, however, has provided evidence for a population of progenitor cells in mammalian forebrain that express properties of differentiated neurons, even though they continue to divide. These neuronal progenitor cells are situated in the rostral migratory stream (RMS), which extends from a specialized portion of the subventricular zone surrounding the anterior tip of the lateral ventricle, referred to as the SVZa, to the middle of the olfactory bulb. As SVZa-derived cells migrate to the olfactory bulb, they undergo cell division, and they never deviate from the RMS. Once they reach their final destinations, they become terminally postmitotic interneurons. This Mini-Review concerns findings from our recent experiments designed to reveal the intrinsic and extrinsic mechanisms governing the proliferation and differentiation of the unique SVZa neuronal progenitor cells. We have investigated the role(s) of cell cycle regulatory proteins, in particular, the cell cycle inhibitor p19(INK4d), in the control of SVZa cell proliferation. Several studies have indicated that cells withdraw from the cell cycle once they express p19(INK4d). To begin to investigate whether p19(INK4d)(+) SVZa-derived cells are postmitotic, we analyzed the pattern of p19(INK4d) expression by the cells of the RMS. A pronounced gradient of p19(INK4d) expression was demonstrated; progressively more cells are p19(INK4d) immunoreactive as the olfactory bulb is approached. In addition, the capacity of p19(INK4d)(+) cells to incorporate bromodeoxyuridine was investigated. From the results of these studies, we conclude that SVZa cells in the RMS can successively down-regulate their expression of p19(INK4d) as they migrate and that they repeatedly exit and reenter the cell cycle while en route to the olfactory bulb. These studies led us to investigate whether bone morphogenetic proteins (BMPs) are involved in the regulation of SVZa cell proliferation and p19(INK4d) expression, because, elsewhere in the CNS, BMPs modulate cell proliferation and influence cell fate decisions. To determine the effects of BMP signaling on SVZa cell proliferation and differentiation, we altered the expression of the BMP receptor Ia (BMPR-Ia) using retrovirally mediated gene transfer. The cells in the SVZa encoding the wild-type BMPR-Ia exit the cell cycle and do not appear to migrate through the RMS. Conversely, both within the SVZa and along the RMS, the majority of SVZa-derived cells encoding a dominant-negative BMPR-Ia gene do not express p19(INK4d). These findings indicate that p19(INK4d) expression is suppressed when BMP signaling is inhibited. Furthermore, SVZa-derived cells with both augmented and inhibited BMP signaling retain their neuronal commitment. Collectively, these studies have revealed that SVZa cell proliferation and differentiation is under the control of several interacting intrinsic and extrinsic factors.

The progenitor cells of the embryonic telencephalon and the neonatal anterior subventricular zone differentially regulate their cell cycle.

For the last 10 years our laboratory has been studying the proliferation, migration and differentiation of neuronal progenitor cells located in the anterior part of the postnatal forebrain subventricular zone (SVZa). SVZa-derived cells possess a number of proliferative characteristics that distinguish them from the other progenitor cells in the central nervous system. This review summarizes our recent findings, in which we compared the pattern of cell cycle inhibitory proteins expressed by the neonatal SVZa to that of telencephalic ventricular zone cells.